Gamma-secretase inhibitors

ABSTRACT

Disclosed, as a γ-secretase inhibitor, is a compound consisting of an amino acid sequence which consists of at least three consecutive amino acids of the amino acid sequence Val-Val-Ile-Ala-Thr-Val-Ile-Val-Ile-Thr-Leu-Val-Met-Leu-Lys-Lys including Leu at position 11, wherein, between the Leu and one or both amino acids located immediately before or after it, the peptide bond, —CO—NH—, is replaced with a hydroxyethylene group, —CHOH—CH 2 —, wherein the N terminus has an alkyloxycarbonyl group based on C1-10 alkyl that may carry phenyl or naphthyl as a substituent group, wherein the C terminus is converted to alkyl ester or alkyl amide based on C1-10 alkyl that may carry phenyl or naphthyl as a substituent group, and wherein the hydrogen atom of the hydroxyl group of the Thr at position 10 may be replaced with a C1-4 hydrophobic group or a Z group, or a pharmaceutically acceptable salt thereof.

TECHNICAL FIELD

The present invention relates to compounds that inhibitgamma-secretease, a splitting enzyme that produces amyloid protein fromthe amyloid-precursor protein. More specifically, the present inventionrelates to those compounds or their pharmaceutically acceptable salts,gamma-secretase inhibitors comprising them, use of the compounds in thescreening of anti-aging drugs or antidementia drugs, as well asantibodies to the compounds.

BACKGROUND ART

Amyloid protein accumulation is a histopathological change occurring incerebral tissues of not only those with Alzheimer's disease or Down'ssyndrome but also those who went through a process of normally aging.The amyloid protein, which consists of from 40 to 42/43 amino acids richin hydrophobic ones, is produced from its precursor, theamyloid-precursor protein (hereinafter referred to as APP), by ahydrolytic cleavage. APP, which consists of 695, 751 or 770 amino acids,is a type 1 membrane protein that spans the membrane once, with itsamino terminal portion exposed outside the cell. The difference in thenumber of its amino acids corresponds to the presence or absence of aso-called Kunitz-type protease inhibitor-active site, which is locatedin the region outside the cell. In neurocytes, a form of APP consistingof 695 amino acids (hereinafter referred to as “APP695”, whose aminoacid sequence is set forth as SEQ ID NO:7), is a dominant one. TheAPP695, consisting of 695 amino acids starting with the methionine atposition 1 and extending up to the asparagine at position 695, is anisoform occurring mainly in neurocytes, and is a type 1 membrane proteinhaving a transmembrane domain (consisting of 24 amino acid extendingfrom the glycine at position 625 to the leucine at position 648) whichspans the cell membrane once. The amyloid protein comprises a shorterform protein molecule consisting of 40 amino acids extending from theaspartic acid at position 597 in the APP695's portion exposed outsidethe cell to the valine at position 636, which is within the cellmembrane, and a longer protein molecule consisting of 42 or 43 aminoacids extending up to the alanine at position 638 or the threonine atposition 639.

On the other hand, a form of APP consisting of 770 amino acids(hereinafter referred to as “APP770”, whose nucleotide sequence in thecoding region is set forth as SEQ ID NO:8 and whose amino acid sequenceas SEQ ID NO:9) is an APP gene product consisting of 770 amino acidsextending from the methionine at position 1 to the asparagine atposition 770 and includes in it an amino acid sequence portion that theAPP695 does not possess (75 amino acids extending from the glutamic acidat position 289 to the lysine at position 363). Apart from this insertedamino acid sequence, it is the molecule having exactly the same aminoacid sequence as those of APP695. Besides the APP695 and the APP770,there is an isoform called APP751, which consists in total of 751 aminoacids, with 19 amino acids lost that extends from the methionine atposition 345 to the lysine at position 363 of the APP770. An amino acidsequence that is commonly inserted into APP751 and APP770 (56 aminoacids extending from the glutamic acid at position 289 to the alanine atposition 344 in the APP770 amino acid sequence) possesses an activity ofthe Kunitz-type protease inhibitor and is thought to be expressed incells other than neurocytes.

Since a hypothesis was experimentally proven that toxicity on neurocytesis one of the physiological activities of the amyloid protein[Yankner,-B-A; Dawes,-L-R; Fisher,-S; Villa-Komaroff,-L;Oster-Granite,-M-L; Neve,-R-L. Neurotoxicity of a fragment of theamyloid precursor associated with Alzheimer's disease. (1989) Science.245(4916): 417-20, and Yankner,-B-A; Duffy,-L-K; Kirschner,-D-A.Neurotrophic and neurotoxic effects of amyloid beta protein: reversal bytachykinin neuropeptides. (1990) Science. 1990 250(4978): 279-82], ithas been assumed as a key molecule of the onset of Alzheimer's disease.Two important steps of reactions must take place for the amyloid proteinto be produced from the amyloid-precursor protein. The first step is thecutting off, by β-secretase, of the portion extending from the aminoterminus of the amyloid protein portion. At the second step, cleavagetakes place at the carboxyl terminus of the amyloid protein by theaction of γ-secretase, resulting in the release of the amyloid protein,with dissociated APP intracellular fragment left behind. According toconventional findings, the cleavage in the second step has been assumedto occur at the gamma (γ) site, which is at the carboxyl terminus of theamyloid protein (between the valine at position 711 and the isoleucineat position 712, between alanine at position 713 and the threonine atposition 714, or between the threonine at position 714 and the valine atposition 715, according to the manner of numbering of amino acidresidues in the APP770). In recent findings, however, it is reportedthat the cleavage occurs at the upsilon (ε) site, downstream by further5-10 amino acid residues (in the direction of the carboxyl terminus),and thus more close to the cytoplasm (between the threonine at position719 and the leucine at position 720, or between the leucine at position720 and the valine at position 721, according to the manner of numberingof amino acid residues in the APP770).

In Alzheimer's disease, neuronal lesions in the brain occur before itsabnormal clinical symptoms will appear, such as disorientation,debilitating memory loss, amnesia, deteriorating judgment, andbehavioral aberration. The neuronal lesions include deposition of theamyloid protein, neurofibrillary tangle, and degenerative cellular loss,among which deposition of the amyloid protein is the earliestpathological event.

The amyloid hypothesis, according to which the production and depositionof amyloid protein does cause the disease, is deemed important withrespect to the progress of Alzheimer's disease. The basis for it hasbeen provided by studies of familial Alzheimer's disease.

There is a speculation yet to be confirmed that γ-secretase is the geneproduct of presenilin-1 [Sherrington,-R; Rogaev,-E-I; Liang,-Y;Rogaeva,-E-A; Levesque,-G; Ikeda,-M; Chi,-H; Lin,-C; Li,-G; Holman,-K;et-al. Cloning of a gene bearing missense mutations in early-onsetfamilial Alzheimer's disease. (1995) Nature, 375(6534), 754-760] andpresenilin-2 [Levy-Lahad,-E; Wasco,-W; Poorkaj,-P; Romano,-D-M;Oshima,-J; Pettingell,-W-H; Yu,-C-E; Jondro,-P-D; Schmidt,-S-D; Wang,-K;et-al. Candidate gene for the chromosome 1 familial Alzheimer's diseaselocus. (1995) Science, 269(5226), 973-977, and Rogaev,-E-I;Sherrington,-R; Rogaeva,-E-A; Levesque,-G; Ikeda,-M; Liang,-Y; Chi,-H;Lin,-C; Holman,-K; Tsuda,-T; et-al. Familial Alzheimer's disease inkindreds with missense mutations in a gene on chromosome 1 related tothe Alzheimer's disease type 3 gene, (1995) Nature 376(6543), 775-778],which were found on the chromosome 14 as causative genes of early-onsetfamilial Alzheimer's disease.

That the APP is also a causative gene of early-onset familialAlzheimer's disease (Goate, A. et al Nature 1991) indicates that aplurality of genes are involved as causative factors of the single classof dementia called Alzheimer's disease.

As mentioned hereinbefore, the amyloid protein comprises two components.One is a shorter amyloid protein component, which starts with asparticacid and ends in the valine at position 40, and the other is a longeramyloid protein component consisting of 42 or 43 amino acids, whichstarts with the same aspartic acid as does the 40 amino-acids componentbut ends in the alanine at position 42 (thus longer by 2 amino acidresidues) or the threonine at position 43 (thus longer by 3 amino acidresidues). The latter longer component is more hydrophobic and thereforestill less soluble. Through addition of the shorter components aroundthe longer one serving as a core, amyloid fiber is formed, which is afibriform structure having the diameter of about 5 to 6 nm.

It has been demonstrated that the production of the longer amyloidprotein increases where a genetic mutation occurs in presenilin-1 orpresenilin-2. It is thus thought that this effect of mutation causeselevated formation of the longer amyloid protein component, whichassumedly determines the threshold level of polymerization of theamyloid protein, and thereby accelerates the pathogenic reaction.

A number of genetic mutations in the APP have been identified, typicalones of which are roughly divided into Sweden mutation (Met670Asn,Lys671Leu, according to the manner of numbering of amino acid residuesin the AP770. The same applies also to mutations described below.),Dutch-type mutation (Glu693Gln), London mutation (Val717Ile), andAustralia mutation (Leu723Pro), which are located just before theposition corresponding to the amino terminus of amyloid protein. Whileproduction of both components of the amyloid protein are found toincrease in Sweden mutation, only the production of the longer amyloidprotein component increases in London mutation and Australia mutation.The effect of Dutch-type mutation is still under discussion and noconclusion has been reached.

There is an allele E4, a risk factor relating to apolipoprotein E.Statistical analyses demonstrated that the onset of Alzheimer's diseasewill accelerated by 8 to 10 yeas where only one allele on the pair ofchromosomes is E4, and by 16 to 20 years where the alleles are E4/E4 onthe both chromosomes [Corder,-E-H; Saunders,-A-M; Strittmatter,-W-J;Schmechel,-D-E; Gaskell,-P-C; Small,-G-W; Roses,-A-D; Haines,-J-L;Pericak-Vance,-M-A., Gene dose of apolipoprotein E type 4 allele and therisk of Alzheimer's disease in late onset families, (1993) Science.261(5123): 921-3].

It is assumed that inhibition of the activity of β-secretase andγ-secretase would suppress the production of amyloid protein, leading totherapeutic drugs that could halt or slow the progress of Alzheimer'sdisease. While a reaction of a-secretase, in addition to β-secretase, isalso involved in the first step of APP hydrolysis, inhibition of thereaction of γ-secretase in the second step is expected to have an effectwith wider spectrum, because that reaction commonly follows either typeof the first step reaction.

Though final identification of γ-secretase is yet to be reached, it isdemonstrated that presenilin-1 is playing an important role. Accordingto animal experiments or cell culture experiments, defunctionalizationof presenilin-1 reportedly caused abnormalities in the development ofcranial nerves or the formation of spinal column, or abnormalities inthe development of lymphocytes. Thus, presenilin-1 is known to have avariety of functions in addition to the one relating the amyloidprotein.

It must be taken account that nonspecific suppression of γ-secretase'sactivity could trigger some severe side effects such as induction ofcancer [Hardy, J., Israel, A. Alzheimer's disease, In search ofgamma-secretase, (1999) Nature. 398(6727) 466-7].

Presently known γ-secretase inhibitors are: inhibitors which arepeptidomimetic compounds based on reports about the γ-site of thesubstrate APP, located at the carboxyl terminus of amyloid proteinportion, on which the enzyme acts [Mori H., Takio K., Ogawara M. &Selkoe D. J. j, Mass spectrometry of purified amyloid b protein inAlzheimer's disease. (1992) J. Biol. Chem. 267, 17082-17086; Roher A. E., Lowenson J. D., Clarke S., Wolkow C., Wang R., Cotter R. J., ReardonI. M., Zurcher-Neely H. A., Heinrikson R. L., Ball M. J., et al.Structural alterations in the peptide backbone of beta-amyloid coreprotein may account for its deposition and stability in Alzheimer'sdisease. (1993) J Biol Chem. 268(5), 3072-3083] and enzyme inhibitorsbased on reports pointing out the importance of aspartic acid activesite [Wolfe M. S., Xia W., Ostaszewski B. L., Diehl T. S., Kimberly W.T., Selkoe D. J. Two transmembrane aspartates in presenilin-1 requiredfor presenilin endoproteolysis and gamma-secretase activity. (1999)Nature 398 (6727), 513-5171.

As for a peptidomimetic compound, DFK-167 is known [Wolfe M. S., CitronM., Diehl T. S., Xia W., Donkor I. C. and Selkoe D. J.: Asubstrate-based difluoro ketone selectively inhibits Alzheimer'sg-secretase activity. (1998) J. Med. Chem., 41(1), 6-9].

As for enzyme inhibitors, compounds screened from known inhibitors arereported. They are, L-685,458, which was made in the process ofdevelopment of drugs for AIDS [Shearman, M. S., Beher, D., Clarke, E.E., Lewis, H. D., Harrison, T., Hunt, P., Nadin, A., Smith, A. L.,Stevenson, G., Castro, J. L. L-685,458, an aspartyl protease transitionstate mimic, is a potent inhibitor of amyloid beta-protein precursorgamma-secretase activity. (2000) Biochemistry 39 (30), 8698-8704] andJLK-6, which is a α-chymotrypsin inhibitor [Nakajima, K., Powers, J. C.,Ashe, B. M., Zimmerman, M. Mapping the extended substrate binding siteof cathepsin G and human leukocyte elastase. Studies with peptidesubstrates related to the alpha 1-protease inhibitor reactive site.(1979) J. Biol. Chem. 254 (10), 4027-32].

Although the peptidomimetic compound-type inhibitors developed based onthe γ-site of the substrate APP and the inhibitors developed based onthe active site of the enzyme are all potent inhibitors of amyloidprotein production, each has significant troubles. First, DFK-167 is aninhibitor that was designed for the γ-site and is a compound totallyindependent from the recent findings on the ε-site, thus it has adifferent target with regard to inhibitor designing. InhibitorsL-685,458 and JLK-6 are not compounds originally developed as specificinhibitors of γ-secretase involved in the production of the amyloidprotein from the APP. Apart from the specificity problem as γ-secretaseinhibitors, there are other problems concerning efficacy at the targettissue.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to provide novel compounds thatinhibit γ-secretase.

Another objective of the present invention is to provide γ-secretaseinhibitor compounds which is focused on the amino acid sequence ofsubstrate APP, not on γ-secretase itself.

A further objective of the present invention is to provide γ-secretaseinhibitor compounds based on the most recent findings about the ε-site.

A still further objective of the present invention is to provide amethod for diagnosis, useful therapeutic drugs and method for treatment,and a method for screening for the development of useful therapeuticdrugs, of Alzheimer's disease and other related diseases. The relateddiseases referred to herein includes diseases in which the amyloidprotein is known or suspected to be directly or indirectly involved asthe pathogen, like Down syndrome, as well as diseases in which amyloidprotein is noted in the neuropathological lesions.

As a result of repeated exercise of ingenuity focused on the ε-site ofAPP (as opposed to the γ-site) in light of the findings of relatedstudies, it was found that inhibition of γ-secretase and suppression ofproduction of amyloid protein are brought about by a class ofpeptide-like compounds which have a similar chemical structure to asequence made of several amino acid including the ε-site of the APP butin which a peptide bond is replaced with a bond stable to the enzymebetween the Leu located at the ε-site (the site where enzymatic cleavagetakes place) and an amino acid located immediately before or after it.The present invention was accomplished based on the finding.

Thus, the present invention provides:

(1) a compound consisting of an amino acid sequence which consists of atleast three consecutive amino acids of the amino acid sequenceVal-Val-Ile-Ala-Thr-Val-Ile-Val-Ile-Thr-Leu-Val-Met-Leu-Lys-Lys-Lys (SEQID NO:1) including Leu at position 11, wherein, between the Leu and oneor both amino acids located immediately before or after it, the peptidebond, —CO—NH—, is replaced with a hydroxyethylene group, —CHOH—CH₂—,while any other inter amino-acid bond is a peptide bond, wherein the Nterminus has an alkyloxycarbonyl group based on C1-10 alkyl that maycarry phenyl or naphthyl as a substituent group, wherein the C terminusis converted to an alkyl ester or alkyl amide based on C1-10 alkyl thatmay carry phenyl or naphthyl as a substituent group, and wherein thehydrogen atom of the hydroxyl group of the Thr at position 10 may bereplaced with a C1-4 hydrophobic group or a Z group, or apharmaceutically acceptable salt thereof,

(2) the compound as described in (1) above, wherein the Leu at position14 of the amino acid sequence is replaced with a hydrophobic amino acidthat may be Ile or with Pro, the Leu at position 11 is replaced with ahydrophobic amino acid that may be Ile, or the Thr at position 10 isreplaced with Ser, or the Ile at position 9 is replaced with ahydrophobic amino acid that may be Leu, or a pharmaceutically acceptablesalt thereof,

(3) a compound consisting of an amino acid sequence which consists of 3,4, 5 or 6 consecutive amino acids of the amino acid sequenceIle-Thr-Leu-Val-Met-Leu (SEQ ID NO:2) including the Leu at position 3,wherein, between the Leu and one or both amino acids located immediatelybefore or after it, the peptide bond, —CO—NH—, is replaced with ahydroxyethylene group, —CHOH—CH₂—, while any other inter amino-acid bondis a peptide bond, wherein the N terminus has an alkyloxycarbonyl groupbased on C1-10 alkyl that may carry phenyl or naphthyl as a substituentgroup, wherein the C terminus is converted to an alkyl ester or alkylamide based on C1-10 alkyl that may carry phenyl or naphthyl as asubstituent group, and wherein the hydrogen atom of the hydroxyl groupof the Thr may be replaced with a C1-4 hydrophobic group or a Z group,or a pharmaceutically acceptable salt thereof,

(4) a compound consisting of the amino acid sequence Leu-Val-Met-Leu(SEQ ID NO:3), wherein, between the Leu at position 1 and the Val atposition 2, the peptide bond, —CO—NH—, is replaced with ahydroxyethylene group, —CHOH—CH₂—, while any other inter amino-acid bondis a peptide bond, wherein the N terminus has an alkyloxycarbonyl groupbased on C1-10 alkyl that may carry phenyl or naphthyl as a substituentgroup, wherein the C terminus is converted to an alkyl ester or alkylamide based on C1-10 alkyl that may carry phenyl or naphthyl as asubstituent group, or a pharmaceutically acceptable salt thereof,

(5) a compound consisting of the amino acid sequence Thr-Leu-Val-Met(SEQ ID NO:4), wherein, between the Thr at position 1 and Leu atposition 2, the peptide bond, —CO—NH—, is replaced with ahydroxyethylene group, —CHOH—CH₂—, while any other inter amino-acid bondis a peptide bond, wherein the N terminus has an alkyloxycarbonyl groupbased on C1-10 alkyl that may carry phenyl or naphthyl as a substituentgroup, wherein the C terminus is converted to an alkyl ester or alkylamide based on C1-10 alkyl that may carry phenyl or naphthyl as asubstituent group, and wherein the hydrogen atom of the hydroxyl groupof the Thr may be replaced with a C1-4 hydrophobic group or a Z group,or a pharmaceutically acceptable salt thereof,

(6) the compound described in (3) above, wherein the Leu locatedimmediately before the Val is replaced with a hydrophobic amino acidthat may be Ile, or the Leu at the N terminus is replaced with ahydrophobic amino acid that may be Ile or with Pro, or apharmaceutically acceptable salt thereof,

(7) the compound described in (4) above, wherein the Leu locatedimmediately before the Val is replaced with a hydrophobic amino acidthat may be Ile, or the Leu at the N terminus is replaced with ahydrophobic amino acid that may be Ile or with Pro, or apharmaceutically acceptable salt thereof,

(8) the compound described in (5) above, wherein the Leu locatedimmediately before the Val is replaced with a hydrophobic amino acidthat may be Ile, or a pharmaceutically acceptable salt thereof,

(9) the compound described in (3) above, wherein the Thr is replacedwith Ser, or a pharmaceutically acceptable salt thereof,

(10) the compound described in (3) above, wherein the Ile is replacedwith a hydrophobic amino acid that may be Leu, or a pharmaceuticallyacceptable salt thereof,

(11) the compound described in (5) above, wherein the Thr is replacedwith Ser, or a pharmaceutically acceptable salt thereof,

(12) the compound of one of (1) to (11) above, wherein thealkyloxycarbonyl group is a Boc group, or a pharmaceutically acceptablesalt thereof,

(13) the compound described in one of (1) to (12) above, wherein apolypeptide consisting of Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg(SEQ ID NO:5) is fused instead of the alkyloxycarbonyl group, or apharmaceutically acceptable salt thereof,

(14) a gamma-secretase inhibitor comprising the compound described inone of (1) to (13) above,

(15) an antibody to the compound described in one of (1) to (13) above,(16) use of the compound described in one of (1) to (13) above as agamma-secretase inhibitor in the screening of an inhibitor of amyloidprotein production.

The compounds of the present invention and acceptable salts thereof maybe used for the treatment of, and for the screening of therapeutic drugsof, Alzheimer's disease and related diseases thereto, such as those inwhich amyloid protein is known or suspected to be directly or indirectlyinvolved as a causative factor of the disease, e.g., Down syndrome, anddiseases in which amyloid protein is observed at a site of aneuropathological lesion. In addition, the antibody of the presentinvention may be used, e.g., for determining the blood levels of thecompound of the present invention administered to a human for treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the first part of a flowchart illustrating the process ofsynthesis.

FIG. 2 shows a part of the flowchart that follows FIG. 1.

FIG. 3 shows a part of the flowchart that follows FIG. 2.

FIG. 4 shows a part of the flowchart that follows FIG. 3.

FIG. 5 shows the last part of the flowchart that follows FIG. 2.

FIG. 6 illustrates the steps for the synthesis of Compound 5.

FIG. 7 shows the first part of a flowchart illustrating the process ofsynthesis of another compound.

FIG. 8 shows a part of the flowchart that follows FIG. 7.

FIG. 9 shows a part of the flowchart that follows FIG. 8.

FIG. 10 shows a part of the flowchart that follows FIG. 9.

FIG. 11 shows a part of the flowchart that follows FIG. 10.

FIG. 12 shows the last part of the flowchart that follows FIG. 11.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, the “allyl” referred to in connection with thealkyloxycarbonyl group is a C1-10, preferably C1-7, and more preferablyC1-5 alkyl. Such an alkyl may be linear or branched and, may be, e.g.,methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, t-butyl, n-pentyl,isopentyl, 2-methylbutyl, 2,2-dimethylpropyl, t-pentyl, hexyl, heptyl,octyl, nonyl, decyl, etc. One of the particularly preferred examples ist-butyl. They may have one or more of their hydrogen atoms replaced witha phenyl group or a naphthyl group. A particularly preferred example ofsuch substituent groups is a benzyloxycarbonyl group.

In the present invention, the “alkyl” referred to in connection with the“conversion to an alkyl ester or alkyl amide” is a C1-10, preferablyC1-7, and more preferably C1-5 alkyl. Such an alkyl may be linear orbranched and, may be, e.g., methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, t-butyl, n-pentyl, isopentyl, 2-methylbutyl,2,2-dimethylpropyl, t-pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.Methyl and t-butyl are particularly preferred examples. They may haveone or more of their hydrogen atoms replaced with a phenyl group or anaphthyl group. One of particularly preferred examples is abenzyloxycarbonyl group.

In the present invention, when the hydrogen atoms of the hydroxyl groupof Thr are replaced with a C1-4 hydrophobic group or with a Z group(i.e., a carbobenzoxy group), examples of the C1-4 hydrophobic groupincludes hydrophobic protective groups such as methyl, ethyl, propyl,n-butyl, sec-butyl, t-butyl, etc.

In the present invention, an “amino acid” means an L-amino acid.

Based on a hypothesis set up in light of the findings from in-depthstudies of APP degradation products, that a stabilized ε-site couldinhibit γ-secretase, the peptidomimetic compounds of the presentinvention was obtained through an attempt of preparing a compound whichhas a structure similar to the ε-site but stabilized to the enzyme, andtheir suppressive effect was confirmed not only on sporadic Alzheimer'sdisease's amyloid production but also on amyloid production involving agene mutation of early-onset familial Alzheimer's disease.

A variety of peptide-like compounds which have an amino acid sequencesimilar to that located around the ε-site of the APP cleaved byγ-secretase but are modified to stabilize the site and have aγ-secretase inhibiting activity, may be used for the purpose of thepresent invention. An example of such a compound is one in which anamino acid residue, —NH—CHR—CO—, in the vicinity of the ε-site isreplaced with a different amino acid residue having a similar propertyregarding to the R (hydrophobicity/hydrophilicity, acidity/basicity,absence ore presence of sulfur atom, presence or absence of a hydroxygroup, etc.), and which has a γ-secretase inhibiting activity. Forexample; Ile, Val, Ala and Gly are amino acids that may be substitutedfor Leu; Leu, Val, Ala and Gly for Ile; Ser for Thr; Ala for Met; Gly,Val, Ile and Leu for Ala; Arg and His for Lys, respectively.Furthermore, the Leu at position 14 of SEQ ID NO:1 (identical to the Leuat position 6 of SEQ ID NO:2 and the Leu at position 4 of SEQ ID NO:3)may be replaced with Pro, for it has been reported that the Aβ42 amyloidprotein component increases in a variant in which the amino acid at thatsite is replaced with Pro (this suggests that such a variant protein hashigher affinity to γ-secretase) [Kwok,-J-B; Li,-Q-X; Hallupp,-M;Whyte,-S; Ames,-D; Beyreuther,-K; Masters,-C-L; Schofield,-P-R, NovelLeu723Pro amyloid precursor protein mutation increases amyloidbeta42(43) peptide levels and induces apoptosis. Ann-Neurol. 2000February; 47(2): 249-53].

As raw materials for synthesizing the compound of the present inventionand methods employed in each step of the synthesis are well known, oneof ordinary skill in the art can prepare an aimed compound by carryingout synthesis, isolation, purification, etc. as desired. It is alsopossible to prepare a polypeptide part of the compound of the presentinvention by application of genetic recombination technology utilizing ahost publicly known per se such as E. coli, yeast, Bacillus subtilis,insect cells, animal cells or plant cells. While chemical synthesis maybe performed, for example, in accordance with the Examples that will bedescribed hereinafter, any other method may be used insofar as it givesthe aimed compound. For example, synthesis may be performed as desiredby employing a combination of well-known methods such asBoc(t-butyloxycarbonyl)-carbonylation reaction, DMSO oxidation, alkalinereaction, acidic reaction, epoxidation, silica gel columnchromatography, alkylation, saponification, reaction by heating,decarboxylation, condensation reaction, reverse-phase columnchromatography, etc. Preferably, a method is employed in which each ofthe structural components of the compound of the present invention issequentially reacted and the yield and the purity of the reactant isassayed at desired steps.

The compounds of the present invention may include such modifications asthose for facilitating synthesis or purification, those for promotingphysical/chemical stability, those for activation in the body relatingto stability and instability or to conditioning to metabolism, and amodification for regulation which causes increase or decrease in theefficiency of transportation to organs including transportation acrossthe blood-brain barrier. The “modification for regulation” referred toherein means a modification with a sequence consisting of the 11 aminoacids, Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (SEQ ID NO:3)[Schwarze, S. R., Ho, A., Vocero-Akbani, A. & Dowdy, S. F. In vivoprotein transduction: Delivery of a biologically active protein into themouse Science 285: 1569-1572]. By having the regulating sequence linkedvia a peptide bond on its N terminus, the compound, thereby facilitatedregarding its transport through the blood brain barrier, becomes capableof reaching more efficiently to a target site in the brain.

Other modifications of the compound of the present invention include,acetylation, acylation, ADP-ribosylation, amidation, attachment of aflavin compound via a covalent bond, attachment of a heme moiety via acovalent bond, attachment of a nucleotide or a nucleotide derivative viaa covalent bond, attachment of a lipid or a lipid derivative via acovalent bond, attachment of phosphatidylinositol via a covalent bond,cross-linking, cyclization, disulfide bond formation, demethylation,formation of a cross-linking covalent bond, cystine formation,pyroglutamate formation, formylation, γ-carboxylation, glycosylation,GPI-anchor formation, hydroxylation, iodization, methylation,myristoylation, oxidation, protein hydrolysis processing,phosphorylation, prenylation, racemization, attachment of a lipid,sulfation, selenoylation, transfer RNA-mediated addition of an aminoacid to the protein such as arginylation, and ubiquitination.

Furthermore, as it will be technically easy to perform structuraladdition, alteration or substitution in order to facilitate detection orpurification of the compound of the present invention or its antibody,as well as to add any other function, any product obtained through suchprocessing will fall within the scope of the present invention. Withinthe scope of the present invention will also fall genetically engineeredproducts modified by addition of, e.g., FLAG-tag, β-galactosidase,alkaline phosphatase, an Fc fragment of immunoglobulins like IgG, andGFP.

[Antibody]

An antibody may be purified using, as an antigen, any compound chosenfrom the compound of the present invention, its derivatives and ordecomposition products. An antigen may be the compound or itsderivative, and consists of, e.g., 20 or less amino acid residues,preferably 5 or less amino acid residues, more preferably 3 and mostpreferably 2 amino acid residues. Purification may also be carried outusing a combination of such antigens. An antigen need not to be thecompound of the present invention itself or its derivative ordecomposition product, but may be one of a structure having, outwardlyexposed, a primary sequence in the vicinity of the APP's ε-site. Theterm “vicinity” referred to herein means a region extending by up to 4amino acids from the ε-site, (which is at either of the 2 positions) inthe direction of the carboxyl terminus.

For preparing an immunospecific antibody to the compound of the presentinvention or its derivative or decomposition compound, a compoundcontaining the aforementioned amino acid sequence in the vicinity of theε-site is preferably used as an antigen. Examples of preferableantibodies include:

(1) an antibody that recognizes the threonine at the carboxyl terminusof Val-Val-Ile-Ala-Thr-Val-Ile-Val-Ile-Thr (SEQ ID NO:10), (2) anantibody that recognizes the leucine located at the carboxyl terminus ofVal-Val-Ile-Ala-Thr-Val-Ile-Val-Ile-Thr-Leu (SEQ ID NO:11),

(3) an antibody that recognizes the leucine at the amino terminus ofLeu-Val-Met-Leu-Lys-Lys-Lys (SEQ ID NO:12),

(4) an antibody that recognizes the valine at the amino terminus ofVal-Met-Leu-Lys-Lys-Lys (SEQ ID NO:13),

(5) an antibody prepared by using an antigen comprising at least twoseries of amino acids including those amino acids recognized in thesequences described in (1) to (4) above.

These antibodies are not limited to a specific class or amount insofaras they immunologically bind to or recognize the site of interest.Binding or recognition of an antibody is determined through publiclyknown antigen-antibody reactions. The term “immunospecific” meanspossessing substantially greater affinity to a compound of interest thanto other relating proteins or compounds known in the prior art.

Using, as an antigen, the compound of the present invention or itsderivative or decomposition product, alone or with a carrier to which itis bound or with which it is mixed, and in the presence or absence of anadjuvant, an antibody is prepared by induction of humoral and/orcellular immune response to the antigen. Otherwise, immuno response maybe induced by immunologically stimulating lymphocytes, or theirprogenitor cells, in culture. Examples of a carrier, on the scope ofwhich no particular limitation is imposed insofar as it has by itself noadverse effect on the host, include, but are not limited to, cellulose,physiological saline, buffered physiological saline, dextrose, water,glycerol, ethanol, polymerized amino acid, albumin and a mixturethereof. Suitable animals used for immunization are mice, rats, rabbits,goats, horses, bovines, etc. A polyclonal antibody may be obtained inthe form of a serum by a publicly known method, or by a method forantibody recovery from a serum. Examples of preferably means includeimmunoaffinity chromatography.

Production of a monoclonal antibody are carried out either by firstharvesting tissues (e.g., the spleen or lymph nodes) containing theantibody activity from animals or harvesting cultured cells immunized asabove, and then introducing transformation into publicly knownperpetually growing cells (e.g., a myeloma line such as P3X63Ag8). Forexample, hybridoma cells prepared from the above antibody producing celland a perpetually growing cells are cloned, and a specific hybridomacell is selected that is producing an antibody that specificallyrecognize the novel compound of the present invention, and the antibodyis collected from the culture medium of the hybridoma. A variety oftechniques for performing this can be enumerated such as those describedas hybridoma method [Kohler G. and Milstein C. (1975) Nature 256,495-497], trioma method [Kozbor et al. Immunology Today (1983) 4: 72],and EBV method [Cole et al. Monoclonal antibodies and cancer therapy,Alan R. Liss, Inc., (1985): 77-96].

The antibody mentioned above may be used for identification, detectionand quantitative determination of the compound of the present inventionor its derivative or decomposition compound, or for preparation andpurification of the compounds utilizing affinity chromatography. Theantibody mentioned above may be converted to a human-type antibody usinga publicly known technique.

Specifically, the compound of the present invention or its derivative ordecomposition compound, and a specific antibody to them having anactivity to enhance the effect of the compound of the present invention,its derivative or decomposition product, are useful as a standardcompound for screening of compounds in search of amyloidprotein-inhibitor drugs, and also as a means for such screening.

By administering its effective amount to a patient of Alzheimer'sdisease or a related disease thereto, in a pharmaceutically acceptablecarrier or without use of a carrier, the compound of the presentinvention is used in order to control the amount of amyloid proteinproduction, thereby preventing or treating those diseases orameliorating their symptoms. The compound of the present invention maybe provided in a suitable preparation form that enhances the efficiencyin the transportation of the compound to the encephalic tissues.

While a species of the compound of the present invention may be usedalone, a plurality of them also may be used in combination. Moreover, aconcomitant use is allowed with other compounds which are beneficial forthe treatment. A preferred form of a pharmaceutical composition forsystemic administration containing the compound of the present inventionis an injection, above all, an intravenous injection. Other routes forinjection, such as subcutaneous, intramuscular and intraperitonealroutes, may also be employed. Another means for systemic administrationis transmucosal or transdermal administration utilizing a penetrationenhancer such as bile salt, fuchsine acid or other surfactants.Furthermore, oral administration is possible when enteric-coatingpreparations or capsules are properly formulated. Topical administrationof such a pharmaceutical composition is also possible, which may be inthe form of a plaster, a paste or a gel.

Examples of assay methods utilizing an antibody to the compound of thepresent invention, its derivatives or decomposition products includeradioimmunoassay, competitive binding assay, high-performance liquidchromatography, Western blot analysis and ELISA, etc. and theircombinations.

EXAMPLES

While the present invention will be described in further detail belowwith reference to examples, it should be noted, however, that thepresent invention is not limited to those examples.

Example 1 Synthesis of the Compound Boc-Leu*-Val-Met-Leu-OMe

Boc-Leu*Val-Met-Leu-OMe (wherein, “*” indicates that the peptide bond“—CO—NH—” between the Leu and the Val is replaced with a hydroxyethylenegroup “—CHOH—CH₂—”) was synthesized as follows according to thesynthetic scheme shown in FIGS. 1-5. The compound was chosen in whichthe configuration around the α-carbon of the hydroxyethylene grouprepresented the R-type. The structure of the compound is shown as theformula (1).

L-leucinol hydrochloric acid (2) (9.87 g, 64.2 mmol) was dissolved in100 ml of acetone and 50 ml of water, and, after addition oftriethylamine (21.5 ml, 128 mmol), refrigerated to −10° C. Boc₂O (20 g,92 mmol) was added dropwise at −10° C. and then stirring was continuedovernight without cooling. Acetone was evaporated and, followingaddition of about 500 ml of 0.1 M hydrochloric acid, extraction wascarried out with 500 ml of ethyl acetate. The organic layer was washedwith 0.1 M hydrochloric acid, water and then saturated saline solutionin the order, and dried with anhydrous sodium sulfate. Following theremoval of the drying agent, the organic layer was concentrated.Purification carried out using a silica gel open column (250 g, ethylacetate/hexane=1/3) gave the aimed compound, Boc-leucinol (3). Yield:12.88 g (92%).

Boc-leucinol (3) (12.88 g, 58.9 mmol) was flushed with toluene and thenpurged with argon gas. It was dissolved in dimethylsulfoxide(dehydrated, 200 ml) and, after addition of triethylamine (22 ml, 158mmol), cooled in a 15° C. water bath. Meanwhile, in another flask,sulfur trioxide-pyridine complex (25.3 g, 159 mmol) was dissolved indehydrated DMSO (100 ml) and stirred for 10 minutes under argon stream.Cooling at 15° C., the sulfur trioxide solution was added dropwise (4minutes) to the solution of Boc-leucinol (3), and, after furtherstirring of 8 minutes, the reaction mixture was poured into 1500 ml ofcold water to terminate the reaction. Following extraction with ethylacetate, the organic layer was washed with 0.1 M hydrochloric acid,water and saturated saline solution in the order, dried with anhydroussodium sulfate, and concentrated after removal of the drying agent.Purification using a silica gel open column (250 g, ethylacetate/hexane=1/5) gave the aimed compound, Boc-Leu-H (aldehyde) (4).Yield: 10.9 g (85.8%).

Sodium hydride (62.6% in mineral oil), following removal of the 0.53 gmineral oil, was purged with argon gas. To this was added DMSO(dehydrated, 50 ml), and the mixture was then heated to 50° C., stirredfor one hour, and, following addition of THF (dehydrated, 50 ml) tothis, cooled on ice. A solution of (CH₃)SI (3.1 g, 15.2 mmol) in DMSO(15 ml) was added dropwise and, one minutes after the start of theaddition, a solution of Boc-Leu-H (aldehyde) (4) (2.0 g, 9.3 mmol) inTHF (10 ml) was added dropwise. After this dropwise addition wascompleted, the mixture was stirred for one hour without cooling. Thereaction mixture was poured into 1000 ml of cold water to terminate thereaction, extracted with ethyl acetate, washed with water and saturatedsaline solution in the order and dried with anhydrous sodium sulfate.After removal of the drying agent by filtration, concentration performedgave 2.2 g of a crude product. Further, reaction of Boc-Leu-H (aldehyde)(4) (4.58 g, 21.3 mmol) under the same condition gave 4.81 g of a crudeproduct (5a and 5b). The crude products from these duplicate reactionswere combined and purified using a silica gel open column (ethylacetate/hexane=1/2). Yield: 5.6 g (80%). This was separated using asilica gel open column (trichloromethane/acetone=100/1), thediastereomers s were purified, and fractions rich in 5a and 5b,respectively, were collected. Determination between the diastereomers 5aand 5b was performed by NMR. The 5a-rich fraction (5a/5b=5/1) was usedin the following process.

Epoxy compound 5a (0.96 g, 4.2 mmol) and diethyl malonate (0.76 ml, 1.2equivalents) were dissolved in 3 ml of ethanol (dehydrated), and, to themixture was added dropwise 2 ml of 20% sodium ethoxide (3 equivalents)while cooling. Cooling then was terminated, and the mixture was stirred20 hours at room temperature, and then poured into a cold aqueoussolution of 10% citric acid to terminate the reaction. Extraction withethyl acetate, washing with water and then saturated saline solution,drying with anhydrous sulfate, removal of the drying agent by filtrationand purification on a silica gel column (ethyl acetate/hexane=1/5) gavecompound 6a. Yield: 1.2 g (83%).

Under argon stream, compound 6a (1.13 g 3.29 mmol) was dissolved in 10ml of dehydrated ethanol. To this were added, under cooling on ice, 20%sodium ethoxide (1.4 ml, 1.2 equivalents) and isopropyl iodide (1.8 ml,3 equivalents). After a five-hour stirring at 60° C., stirring wascontinued overnight at room temperature, and then 7 hours at 60° C. onthe following day. The reaction was terminated by pouring the mixtureinto a 10% cold aqueous solution of citric acid. Extraction with ethylacetate, washing of the organic layer, drying with sodium sulfate,removal of the drying agent by filtration and evaporation of the solventgave a crude product. Purification using a silica gel column (20 g,ethyl acetate/hexane=1/3) gave the aimed compound 7. Yield: 0.8 g (63%).

Lactone-ester (7) (0.78 g, 2.0 mmol) was dissolved in about 10 ml ofdioxane. Cooling on ice, 1 N sodium hydroxide (4 ml) was added dropwiseand the mixture was stirred at room temperature. After a 1.5-hourstirring, dioxane was evaporated, and the residue was poured into a cold0.2 N HCl aqueous solution, and extracted with ethyl acetate. Theorganic layer was washed with saturated saline solution, and dried oversodium sulfate. Filtered and concentrated, this gave a crude product oflactone-carboxylic acid (8). This was directly dissolved in toluene, andthen heated on a 95° C. oil bath (3 hours), allowed to cool downovernight, and heated again 95° C. on the following day. One and a halfhours later, toluene was evaporated and purification was performed usinga silica gel column (ethyl acetate/hexane=1/5). Of two spatiallyapproximate spots on the TLC of the products, 0.4 g of a compound (9a)corresponding to the lower spot and 0.2 g of a compound (9b)corresponding to the upper spot were obtained. Yield: 63% and 32%,respectively.

Compound 9a (0.37 g, 1.18 mmol) was dissolved in 2.5 ml of dioxane, and1 N sodium hydroxide aqueous solution was added dropwise underice-cooling, and stirred for 2 hours at room temperature. The reactionwas then terminated by addition of a 20% aqueous solution of citric acidand extracted with ethyl acetate. The organic layer was washed withwater and then with a saturated saline solution, dried with sodiumsulfate, filtered and concentrated, thus giving hydroxycarboxylic acid(10). This was dissolved in anhydrous DMF, and after successive additionof imidazole (1.7 g, 25 mmol), t-butyldimethylsilyl chloride (1.8 g, 12mmol) and DMAP (25 mg, 0.2 mmol), stirred overnight at room temperature.To this was added about 1 ml of methanol, and stirred for 30 minutes tokill the reagents. The mixture was poured into a 20% citric acidsolution, and extracted with ethyl acetate. The organic layer was washedwith water and then with saturated saline solution, dried with sodiumsulfate, filtered and concentrated. This (disilyl compound) wasdissolved in acetic acid and stirred for 4 hours at room temperature(cleavage of the silyl ester was confirmed by TLC and MS of the reactionmixture). After removal of acetic acid under reduced pressure,purification using a silica gel column (ethyl acetate/hexane=1/2) gavecompound 11. Yield: 0.5 g (96%).

[Synthesis of an Isomer of Compound 11]

Starting with 9b, the same process as above gave an isomer of silylcompound 11. Yield: 0.2 g (88%).

[Synthesis of Hydrochloric Acid Salt of Leu-OMe]

Anhydrous methanol (150 ml) was placed in a flask and cooled down (−15°C.). To this was added SOCl₂ (40 ml) dropwise, and 10 minutes laterL-leucine powder was directly added, and, with the cooling terminated,stirred at room temperature. About ten minutes later, further 100 ml ofanhydrous methanol was added and stirring was continued overnight.Twenty hours later, the solvent was evaporated under reduced pressure,and, after flushing with methanol twice, diethyl ether was added to thecrystalline residue. Precipitates were collected by filtration and driedover sodium hydroxide under reduced pressure. Yield: 24.23 g (87.8%).

[Synthesis of Boc-Met-Leu-OMe]

HCl, Leu-OMe (5.3 g, 29.2 mmol), Boc-Met (7.64 g, 30.6 mmol) and1-hydroxy-1H-benzotriazole (HOBt) (4.34 g, 32 mmol) were weighed into a500-ml flask, and dissolved in DMF. While cooling, WSCD (5.88 ml, 32mmol) wad added dropwise. Two hours later, the mixture was poured into acold 2% sodium bicarbonate aqueous solution, and precipitating crystalswere collected by filtration and washed with water. They were dissolvedin 300 ml of ethyl acetate and washed with 0.2 N hydrochloric acid,water and then saturated saline solution, and dried with sodium sulfate.After evaporation of the solvent, precipitated crystals were collectedby filtration using hexane. Dried over phosphorus pentoxide, the aimedcompound (13) was obtained. Yield: 10.4 g (94.6%).

[Process for Boc Removal]

While cooling, TFA was added to Boc-Met-Leu-OMe (1.09 g, 2.89 mmol), and10 minutes later, the cooling was terminated and stirring was continuedfor 50 minutes at room temperature. TFA was evaporated and 4.9 Nhydrochloric acid/dioxane (0.71 ml) was added. After several timesdecantation with hexane, concentration to dryness under reduced pressuregave a solid. Yield: 0.8 g (90% as hydrochloride).

[Condensation Reaction]

To the above hydrochloride (0.29 g, 0.93 mmol) were added compound 11(0.34 g, 0.76 mmol) and HOOBt (150 mg, 0.92 mmol) and the mixture wasdissolved in DMF (10 ml). While cooling, WSCD (170 μl, 0.93 mmol) wasadded dropwise, and, with the cooling terminated, stirred was continuedat room temperature. Twenty hours later, the mixture was poured into acold 2% aqueous solution of sodium bicarbonate to terminate thereaction. The mixture was extracted with ethyl acetate and washed with a10% citric acid, water and saline solution. After drying with sodiumsulfate, concentration and purification using a silica gel column (ethylacetate/hexane=1/4) gave compound 14. Yield: 0.5 g (93%).

The protected compound 14 (0.5 g, 15-02011221) was dissolved anhydrousTHF. This was desilylated by addition of TBAF-3H₂O (330 mg, 1.05 mmol),followed by a 24-hour stirring at room temperature. After evaporation ofTHF, purification using a silica gel column (ethylacetate/hexane=1/1-1/2) gave 0.25 g of a crude product of aimed compound(1), with 0.14 g of the starting material recovered. Purification byreverse-phase HPLC (YMC—ODS, acetonitrile/water/0.1% TFA), andlyophilization gave the aimed compound. Yield: 106 mg (25%). ESI-MS:590.3(M+H, Calculated value 590.38), 612.3 (M+Na).

[Synthesis of an Isomer of Compound 1]

Using the aforementioned isomer (0.18 g) of compound 11, condensation,removal of the protective group, and purification in the same mannergave 46 mg of an isomer of compound 1. ESI-MS: 590.3(M+H, Calculatedvalue 590.38), 612.3(M+Na).

Example 2 Examination of Solubility

The compound synthesized in Example 1 was examined for its solubility,visually as well as by microscopy. The results are shown in the tablebelow. TABLE 1 Solubility of the Compound Concentration ConcentrationConcentration Solvent (10 ng/ml) (1 μg/ml) (100 μg/ml) Physiological ++/− − saline Phosphate buffer + +/− − (pH 7.0) DMEM medium + +/− −containing 10% fetal bovine serum DMSO + + ++, dissolved; +/−, nearly dissolved, −, not dissolved

Example 3 Determination of Inhibitory Activity

The human APP695 gene used in this example (whose nucleotide sequence inits coding region is shown as SEQ ID NO:6) was what had been screenedfrom a cDNA library prepared from the dead brain of a normal human by aconventional method, and its entire nucleotide sequence was confirmed bya conventional method. Briefly, about 1 g of frozen cerebral tissue wasphenol-treated and the supernatant aqueous solution was collected. Afterrepeating this process, mRNA was precipitated by addition of ethanol.After dissolving in 20 mM tris-HCl, pH 8., 0.1 mM EDTA, cDNA wassynthesized with reverse transcriptase using oligo-dT primers and themRNA as a template. After remaining RNA was digested with RNase, usingthus obtained single-stranded cDNA as a template, double-stranded cDNAwas synthesized with DNA polymerase. The double-stranded cDNA was thenblunt-ended with T4 DNA polymerase. Following introduction ofmethylation into a EcoR1 restriction site using EcoR1 methylase, anEcoR1 linker adapter was ligated using ligase. After removal ofunreacted free adapter by gel filtration, ligation to λgt11 vector waseffected using DNA ligase, and thus ligated product was incorporatedinto λ-phage using a packaging kit. E. coli cells in agar plates wereinfected with the λ-phage thus obtained, and the solution containingthus proliferated phage was used as cDNA library. For APP cDNA cloning,oligonucleotides that were anticipated based on the amyloid protein wereradio-labeled using γ³²P-ATP and DNA kinase to provide the labelednucleotides as probes. 10⁶ pfu (plaque forming units) of phage wereallowed to form plaques on agar plates, and brought into contact with anylon membrane. The phage DNAs that stuck to the membrane weredenaturated to single-stranded DNAs by alkali treatment of the nylonmembrane, and the membrane then was neutralized, to which, afterincubation in a solution containing salmon sperm DNAs, the ³²Pradio-labeled nucleotide probes were added and allowed to hybridize for12 hours. At this stage, where aimed APP gene incorporated to the phageand one of the oligonucleotides were mutually complementary in theirsequences, they would form a double-stranded DNA. The nylon membrane wasthen exposed to X-ray film, and plaques on the agar plates correspondingto spots of exposure were identified with phages carrying APP gene(fragment). By repeating this process, a single phage was finallyobtained. The nucleotide sequence of the APP gene incorporated into thephage was determined by publicly known Sanger method.

APP gene was efficiently introduced from outside into COS cells, whichis a cultured cell line of simian kidney's origin, using LipofectaminePlus reagent (Invitrogen K.K.) according to its protocol. Briefly, 1 μgof APP gene was dissolved in 100 μl of OPTI-MEM, mixed with 6 μl of Plusreagent and left to stand for 15 minutes at room temperature. Inparallel, 4 μl of Lipofectamine dissolved in 100 μl of OPTI-MEM mediumwas prepared. This was then mixed with the APP gene solution, and leftto stand for further 15 minutes. Six-well culture plates were providedin advance in each well of which was placed 800 μl of culture that hadbeen prepared by culturing COS cells at 75% to 80% cell density in aDMEM medium supplemented with 10% fetal bovine serum, and then replacingthe medium with OPTI-MEM medium. Two hundred pI of the reaction mixturecontaining the gene were added to each culture well. Twelve hours afterintroduction of the gene, the medium was changed from the OPTI-MEMmedium containing the introduced gene to a 10% fetal bovineserum-supplemented DMEM medium free of the introduced gene. To thisculture, the compound obtained in Example 1 was added. After 36 to 48hours, culture supernatant was collected, transferred to centrifugingtubes, and centrifuged at 10,000 r.p.m. at room temperature for 5minutes. The supernatant was used as a sample containing amyloidprotein. The shorter and the longer amyloid protein components containedin each sample can be separately determined using a commerciallyavailable ELISA kit for amyloid protein determination (KHB3441: SignalSelect™ Human ⊖ Amyloid1-42 ELISA Kit, or KHB3481: Signal Select™ Humanβ Amyloid1-40 ELISA Kit, BioSource International, Inc. CA, USA). ThoseELISA kits for amyloid determination included 96-well ELISA platescoated with a primary antibody to these two types of amyloid proteins.Following eliminating non-specific antigen-antibody reactions byallowing a usual non-specific blocking reaction, a sample mediumcontaining the amyloid protein to be measured was reacted. Then, afterusual washing, one of secondary antibodies was added. The secondaryantibodies consisted of two deferent antibodies that can distinguishbetween the two types of amyloid proteins, the one is the shorteramyloid protein and the other is the longer amyloid antibody, without nocross reactivity. The difference between the two amyloid proteins is theabsence or presence of the two amino acid residues (isoleucine andalanine) at their carboxyl terminus. The antibody that detects theformer is considered to recognize valine at the carboxyl terminus andthe antibody that detects the latter is thought to recognize thealanine.

The results of the amyloid protein determination using the kit are shownin the following tables. TABLE 2 Compound's Inhibitory Activity-1 DMSOInhibitor (100 μM) Ratio of Ratio of Aβ42(43) Aβ40 Aβ42(43)/ Aβ42(43)Aβ40 Aβ42(43)/ (pg/ml) (pg/ml) Aβ40 (pg/ml) (pg/ml) Aβ40 1 Mock 0 0 — 00 — treatment 2 Wild-type 1415 9656 0.147 415 218 1.90 APP695 3 London-2845 6623 0.430 855 151 5.66 type APP695 4 Sweden- 2503 17002 0.147 13111726 0.76 type APP695

TABLE 3 Compound's Inhibitory Activity-2 Inhibition rate (%) Aβ42(43)Aβ42 Total Aβ 1 Mock — — — treatment 2 Wild-type 71 98 98 APP695 3London- 70 98 89 type APP695 4 Sweden- 48 90 84 type APP695

As evident from Tables 1 and 2, the inhibitor exhibited an inhibitoryactivities of about 71% for Aβ42(43) production and nearly 100%, i.e.,about 98% for Aβ40 production. About 98% inhibition was achieved even onthe total amyloid protein. Thus, it was revealed that the presentinhibitor has an effective activity.

Although it is a rare class of disease comprising only several % oftotal Alzheimer's disease cases, inhibitory activity on amyloid proteinproduction in familial early-onset Alzheimer's disease was alsoexamined. According to the results, nearly the same level of inhibitoryactivity as that noted on the wild-type sequence was observed onVal717Ile mutation of amyloid protein, called London mutation. On theother hand, on a double mutation discovered in Sweden, Met670Arn andLys671Leu, inhibitory activity was about 90% for Aβ40 and about 48% forAβ42(43). On each mutation of the familial disease, more than about 80%inhibition was noted for total amyloid production.

Example 4 Determination of Inhibitory Activity

In Examination 3, a full sized human APP gene was employed. The amyloidprotein is formed through proteolysis consisting of the first-stepcleavage of the APP by α- or β-secretase and the second-step cleavage byγ-secretase. Besides γ-secretase inhibition, the inhibition in thefirst-step could lead to suppression of amyloid protein synthesis. Inorder to directly demonstrate that the inhibitory effect of the compoundof the present invention is due to inhibition of γ-secretase (but notdue to inhibition of β-secretase), an experiment was carried out in thesame manner as in Example 4 except that an APP artificial fragment(referred to as C100), which lacked a polypeptide portion that is to becut off in the first stage, was used instead of APP695, and amyloidprotein thus produced was measured. The results are shown in thefollowing table. TABLE 4 Compound' Inhibitory Activity-3 DMSO TotalInhibitor (100 μM) Inhibition rate (%) Aβ42(43) Aβ40 Aβ Aβ42(43) Aβ40Total Aβ42 Total (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) Aβ (43) Aβ40 Aβ1 C100 127 944 1071 62 100 162 51 89 85

In the experiment using a C-100 APP (wild type), nearly the sameinhibitory activity was observed as was in the Sweden-type mutation,i.e., about 90% inhibition for Aβ40 and about 50% inhibition forAβ42(43). In each case where a familial disease-type mutation ispresent, more than about 80% of inhibition was observed on totalproduction of amyloid protein.

Example 5 Synthesis of the Compound t-But-OCO-Thr*-Leu-Val-Met-NH-Bzl

T-But-OCO-Thr*-Leu-Val-Met-NH-Bzl (wherein, “*” indicates that thepeptide bond “—CO—NH—” between the Thr and the Leu is replaced with ahydroxyethylene group “—CHOH—CH₂—” and that the hydroxyl group of theThr is converted to t-butyl ether) was synthesized as follows accordingto the synthetic scheme shown in FIGS. 7-12. Both R- and S-typecompounds were synthesized with regard to the configuration aroundα-carbon of the hydroxyethylene group of the compounds. Out of these,the R-type compound is shown in the formula (1a).

Synthesis of Aldehyde 3′:

DCHA salt of 2′ (49.5 g, 100.88 mmol) (Bachem 123400) was dissolved inethyl acetate (500 ml), and demineralized with a 20% citric acid aqueoussolution. The organic layer was washed with water (three times) and thensaturated saline solution, dried with Na₂SO₄, and, after removal of thedrying agent by filtration, concentrated to give a oily residue. Theresidue was then dissolved in anhydrous THF (500 ml), cooled to −15° C.,and isobutyl chlorocarbonate (15.7 ml, 121 mmol) was added dropwise.After a 10-minute stirring at −15° C., NaBH₄ was added and, afterstirring was continued for 10 minutes at −15° C., then cooled down to−50° C. and methanol (500 ml) was added dropwise. Following dropwiseaddition of 1 N hydrochloric acid (200 ml), the mixture was stirred for30 minutes while letting it return to room temperature. Afterevaporation of the solvent, extraction with ethyl acetate, washing with0.2 N hydrochloric acid, water (three times) and saturated purifiedwater in the order, drying with Na₂SO₄ and evaporation of the solventgave an alcohol (39 g). Thirty-nine g of this alcohol was divided intothree portions and 13 g each were subjected to oxidizing reaction at atime. Briefly, Z-Thr(tBu)-ol (13 g, 33 mmol), after subjected twice tothe process of dissolution in toluene and evaporation of the solvent,was dissolved in anhydrous DMSO (120 ml), and, after addition oftriethylamine (13.9 ml, 100 mmol), cooled in a water bath at 15° C.Sulfur trioxide-pyridine complex (15.9 g, 100 mmol) was dissolved inDMSO (70 ml) in another flask, and the solution was added dropwise tothe Z-Thr(tBu)-ol solution over 2 minutes. After 8 minutes of stirringin the 15° C. water bath, the reaction was terminated by pouring themixture into ice-cold water. Extraction was carried out with ethylacetate. The remaining portions of the alcohol were subjected to thereaction in the same manner. All the ethyl acetate extract was combined,washed with water and then saturated saline solution, dried with Na₂SO₄,concentrated, and purified using a silica gel column (AcOEt/Hex=1/4) togive the aimed aldehyde (3′). Yield: 24.1 g (81% from the carboxylicacid).

Synthesis of Epoxy Compound (4′):

The aldehyde (3′), divided into two halves, was subjected to reaction intwice. DMSO (anhydrous, 200 ml) was added to 1.9 g sodium hydride (62.6%in oil), and heated to 50° C. and stirred for one hour. To thus formeddimethylsulfinyl anion solution was added THF (anhydrous, 200 ml) andthe mixture was cooed on ice. To this was added a DMSO solution (50 ml)of (CH₃)SI (10 g, 49 mmol) dropwise (over 20 seconds), and one minuteafter the start of the dropwise addition, a THF solution (510 ml) ofZ-Thr(tBu)-H (12.0 g, 40.9 mmol) was added dropwise (over one minute).After the dropwise addition was finished, stirring was continued at roomtemperature (20° C.) for one hour and the reaction was terminated bypouring the mixture into 1500 ml of cold water. Extraction with ethylacetate, washing with water and saturated saline solution in the order,drying with anhydrous sodium sulfate and concentration gave 10 g ofcrude product. A second series of reactions carried out in the samemanner gave 10 g of crude product. The products of these two series ofreactions were combined, purified using a silica gel column(AcOEt/Hex=1/2), and further purified using a silica gel column(AcOEt/Hex=1/4) to give the aimed epoxy compound (4′). 6.3 g (25%).

Synthesis of Compound (5):

The epoxy compound (4) (6.3 g, 20.5 mmol) was dissolved in anhydrousethanol (10 ml) and, after addition of diethyl malonate (3.8 ml, 25mmol), cooled on ice. While ice-cooling, 20% NaOEt (9.6 ml, 24.5 mmol)was added dropwise, and, after a 3-hour stirring, a 10% citric acidaqueous solution was added. Extraction was performed with ethyl acetate,and the ethyl acetate layer was washed with water and then withsaturated saline solution, dried with Na₂SO₄, concentrated, and purifiedusing a silica gel column (AcOEt/Hex=1/2) to give (5′). Yield: 7.66 g(89%).

Synthesis of Compound (6′):

The ethyl ester 5′ (7.38 g, 17.5 mmol) was dissolved in methanol (35ml), and to the mixture was added 1 N NaOH (35 ml) dropwise whileice-cooling. After stirring for 2 hours at room temperature, the mixturewas poured into a 20% citric acid aqueous solution and extracted withethyl acetate. The organic layer was washed with water and saturatedsaline solution, dried with sodium sulfate, and concentrated. This wasthen dissolved in toluene (50 ml) and heated on an oil bath to 90° C.,and, 6 hours later, toluene was evaporated under reduced pressure.Purification using a silica gel column (AcOEt/Hex=1/2) gave compound(6). Yield: 0.95 g (15.5% from the ethyl ester).

Synthesis of Compound (7′):

The lactone compound (6′) (0.95 g, 2.72 mmol) was dissolved in anhydrousTHF (20 ml) and cooled to −78° C. and 1.1 N LiHMDS (5.4 ml) was addeddropwise. After 30 minutes of stirring at −78° C.,3-bromo-2-methyl-propene (550 μl, 5.45 mmol) was added and stirring wascontinued at −78° C. for 40 minutes. The reaction was terminated byaddition of saturated ammonium chloride aqueous solution and extractionwas performed with ethyl acetate. Washing with saturated salinesolution, drying with Na₂SO₄ and purification using a silica gel columngave 0.29 g of the aimed alkene (7′), with recovery of 0.17 of thestarting material. The recovered starting material was subjected to thereaction again under the same equivalence conditions, worked up,purified, and the product was combined with the product obtained by thefirst reaction. Yield: 0.37 g (34%).

Synthesis of Compound (8′):

The alkene (7′) (0.37 g, 0.92 mmol) was dissolved in methanol (10 ml),and 5% Pd-C was added and hydrogen gas introduced to perform catalyticreduction. Four hours later, nitrogen gas was introduced to terminatethe reaction. After removal of the catalyst by filtration and thenconcentration, the residue was dissolved in methanol (20 ml), and tothis were added triethylamine (TEA) (256 μl, 1.84 mmol) and Boc₂O (400mg, 1.84 mmol) in the order, and the mixture was stirred overnight atroom temperature. After concentration, purification using a silica gelcolumn (AcOEt/Hex=1/4) gave the aimed compound (8′). Yield: 0.48 g.

Synthesis of Compounds (9′), (10′, 1′b, 11′, 1′a):

The lactone (8′) was saponified with sodium hydroxide in methanol. Afterprecipitated with acid and extracted, this was converted to a silylether with alkylsilylchloride, imidazole and DMAP in DMF, and purifiedusing a silica gel column to give carboxylic acid (9′). The carboxylicacid (9′) and an amino moiety (dipeptide H-Val-Met-NHBzl) were condensedin DMF solvent using EDC and HOBt. After the reaction, workup includingextraction, washing, etc. and purification using a silica gel columngave (10′). The silyl ether (10) was TBAF-treated in THF solvent. Afterthe reaction, concentration and combined purification using a silica gelcolumn and ODS-HPLC gave (1′b: TLVM-2). This was subjected to Swernoxidation in DMSO using sulfur trioxide-pyridine complex andtriethylamine. After workup including extraction, removal of byproductssuch as sulfuric ester by ODS-HPLC separation gave ketone compound(11′). The ketone (11′) was dissolved in methanol and reduced with NaBH₄to give an alcohol. This reducing reaction gave a mixture of (1′a) and(1′b). As they were readily separable by ODS-HPLC, they were purified byHPLC separation, thus giving (1′a: TLVM-2).

By the way, staring with this compound, beginning with removal andreplacement of its protecting group with a Z group, or, instead, throughreplacement of the t-butoxycarbonyl group with a Z group at a properstage in the course of synthesis, similar flow of reactions and removalof the t-butyl group will give the compound shown below.

Example 6 Determination of Inhibitory Activity-3

The compounds (1′a) and (1′b) obtained above were measured forinhibitory activity by the same method as in Example 3. As the systemfor evaluation, however, IMR32, a neural system cell line of humanorigin, was used after introducing to it a human APP695 gene into whichthe Sweden mutation had been incorporated. TABLE 5 Aβ40 (pg/ml)Inhibition rate (%) Compound 1'a 100 μM 0 100  10 μM 31 73 Compound 1'b100 μM 26 78  10 μM 72 38

INDUSTRIAL APPLICABILITY

The present invention provides novel peptidomimetic compounds relatingto the pathology of Alzheimer's disease and related diseases thereto.Provision of novel pharmaceutical compositions, as well as means fordiagnosis that utilize the characteristics of the compounds, is usefulin the clinical and medical field of diseases to which the presentcompounds relate, in particular, Alzheimer's disease and relateddiseases thereto.

1. A compound consisting of an amino acid sequence which consists of atleast three consecutive amino acids of the amino acid sequenceVal-Val-Ile-Ala-Thr-Val-Ile-Val-Ile-Thr-Leu-Val-Met-Leu-Lys-Lys-Lys (SEQID NO:1) including Leu at position 11, wherein, between the Leu and oneor both amino acids located immediately before or after it, the peptidebond, —CO—NH—, is replaced with a hydroxyethylene group, —CHOH—CH₂—,while any other inter amino-acid bond is a peptide bond, wherein the Nterminus has an alkyloxycarbonyl group based on C1-10 alkyl that maycarry phenyl or naphthyl as a substituent group, wherein the C terminusis converted to an alkyl ester or alkyl amide based on C1-10 alkyl thatmay carry phenyl or naphthyl as a substituent group, and wherein thehydrogen atom of the hydroxyl group of the Thr at position 10 may bereplaced with a C1-4 hydrophobic group or a Z group, or apharmaceutically acceptable salt thereof.
 2. The compound of claim 1,wherein the Leu at position 14 of the amino acid sequence is replacedwith a hydrophobic amino acid that may be Ile or with Pro, the Leu atposition 11 is replaced with a hydrophobic amino acid that may be Ile,or the Thr at position 10 is replaced with Ser, or the Ile at position 9is replaced with a hydrophobic amino acid that may be Leu, or apharmaceutically acceptable salt thereof.
 3. A compound consisting of anamino acid sequence which consists of 3, 4, 5 or 6 consecutive aminoacids of the amino acid sequence Ile-Thr-Leu-Val-Met-Leu (SEQ ID NO:2)including the Leu at position 3, wherein, between the Leu and one orboth amino acids located immediately before or after it, the peptidebond, —CO—NH—, is replaced with a hydroxyethylene group, —CHOH—CH₂—,while any other inter amino-acid bond is a peptide bond, wherein the Nterminus has an alkyloxycarbonyl group based on C1-10 alkyl that maycarry phenyl or naphthyl as a substituent group, wherein the C terminusis converted to an alkyl ester or alkyl amide based on C1-10 alkyl thatmay carry phenyl or naphthyl as a substituent group, and wherein thehydrogen atom of the hydroxyl group of the Thr may be replaced with aC1-4 hydrophobic group or a Z group, or a pharmaceutically acceptablesalt thereof.
 4. A compound consisting of the amino acid sequenceLeu-Val-Met-Leu (SEQ ID NO:3), wherein, between the Leu at position 1and the Val at position 2, the peptide bond, —CO—NH—, is replaced with ahydroxyethylene group, —CHOH—CH₂—, while any other inter amino-acid bondis a peptide bond, wherein the N terminus has an alkyloxycarbonyl groupbased on C1-10 alkyl that may carry phenyl or naphthyl as a substituentgroup, wherein the C terminus is converted to an alkyl ester or alkylamide based on C1-10 alkyl that may carry phenyl or naphthyl as asubstituent group, or a pharmaceutically acceptable salt thereof.
 5. Acompound consisting of the amino acid sequence Thr-Leu-Val-Met (SEQ IDNO:4), wherein, between the Thr at position 1 and Leu at position 2, thepeptide bond, —CO—NH—, is replaced with a hydroxyethylene group,—CHOH—CH₂—, while any other inter amino-acid bond is a peptide bond,wherein the N terminus has an alkyloxycarbonyl group based on C1-10alkyl that may carry phenyl or naphthyl as a substituent group, whereinthe C terminus is converted to an alkyl ester or alkyl amide based onC1-10 alkyl that may carry phenyl or naphthyl as a substituent group,and wherein the hydrogen atom of the hydroxyl group of the Thr may bereplaced with a C1-4 hydrophobic group or a Z group, or apharmaceutically acceptable salt thereof.
 6. The compound of claim 3,wherein the Leu located immediately before the Val is replaced with ahydrophobic amino acid that may be Ile, or the Leu at the N terminus isreplaced with a hydrophobic amino acid that may be Ile or with Pro, or apharmaceutically acceptable salt thereof.
 7. The compound of claim 4,wherein the Leu located immediately before the Val is replaced with ahydrophobic amino acid that may be Ile, or the Leu at the N terminus isreplaced with a hydrophobic amino acid that may be Ile or with Pro, or apharmaceutically acceptable salt thereof.
 8. The compound of claim 5,wherein the Leu located immediately before the Val is replaced with ahydrophobic amino acid that may be Ile, or a pharmaceutically acceptablesalt thereof.
 9. The compound of claim 3, wherein the Thr is replacedwith Ser, or a pharmaceutically acceptable salt thereof.
 10. Thecompound of claim 3, wherein the Ile is replaced with a hydrophobicamino acid that may be Leu, or a pharmaceutically acceptable saltthereof.
 11. The compound of claim 5, wherein the Thr is replaced withSer, or a pharmaceutically acceptable salt thereof.
 12. The compound ofone of claims 1 to 11, wherein the alkyloxycarbonyl group is a Bocgroup, or a pharmaceutically acceptable salt thereof.
 13. The compoundof one of claims 1 to 12, wherein a polypeptide consisting ofTyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (SEQ ID NO:5) is fusedinstead of the alkyloxycarbonyl group, or a pharmaceutically acceptablesalt thereof.
 14. A gamma-secretase inhibitor comprising the compound ofone of claims 1 to
 13. 15. An antibody to the compound of one of claims1 to
 13. 16. Use of the compound of one of claims 1 to 13 as agamma-secretase inhibitor in the screening of inhibitors of amyloidprotein production.